New chemical method of viral inactivation for vaccine development based on membrane fusion inhibition

ABSTRACTThe core, conserved function of the herpesvirus gH/gL is to promote gB-mediated membrane fusion during entry, although the mechanism is poorly understood. The human cytomegalovirus (HCMV) gH/gL can exist as either the gH/gL/gO trimer or the gH/gL/UL128/UL130/UL131 (gH/gL/UL128-131) pentamer. One model suggests that gH/gL/gO provides the core fusion role during entry into all cells within the broad tropism of HCMV, whereas gH/gL/UL128-131 acts at an earlier stage, by a distinct receptor-binding mechanism to enhance infection of select cell types, such as epithelial cells, endothelial cells, and monocytes/macrophages. To further study the distinct functions of these complexes, mutants with individual charged cluster-to-alanine (CCTA) mutations of gH and gL were combined to generate a library of 80 mutant gH/gL heterodimers. The majority of the mutant gH/gL complexes were unable to facilitate gB-mediated membrane fusion in transient-expression cell-cell fusion experiments. In contrast, these mutants supported the formation of gH/gL/UL128-131 complexes that could block HCMV infection in receptor interference experiments. These results suggest that receptor interactions with gH/gL/UL128-131 involve surfaces contained on the UL128-131 proteins but not on gH/gL. gH/gL/UL128-131 receptor interference could be blocked with anti-gH antibodies, suggesting that interference is a cell surface phenomenon and that anti-gH antibodies can block gH/gL/UL128-131 in a manner that is distinct from that for gH/gL/gO.IMPORTANCEInterest in the gH/gL complexes of HCMV (especially gH/gL/UL128-131) as vaccine targets has far outpaced our understanding of the mechanism by which they facilitate entry and contribute to broad cellular tropism. For Epstein-Barr virus (EBV), gH/gL and gH/gL/gp42 are both capable of promoting gB fusion for entry into epithelial cells and B cells, respectively. In contrast, HCMV gH/gL/gO appears to be the sole fusion cofactor that promotes gB fusion activity, whereas gH/gL/UL128-131 expands cell tropism through a distinct yet unknown mechanism. This study suggests that the surfaces of HCMV gH/gL are critical for promoting gB fusion but are dispensable for gH/gL/UL128-131 receptor interaction. This underscores the importance of gH/gL/gO in HCMV entry into all cell types and reaffirms the complex as a candidate target for vaccine development. The two functionally distinct forms of gH/gL present in HCMV make for a useful model with which to study the fundamental mechanisms by which herpesvirus gH/gL regulates gB fusion.

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Viral Inactivation Based on Inhibition of Membrane Fusion: Understanding the Role of Histidine Protonation to Develop New Viral Vaccines

Protein and Peptide Letters ◽

10.2174/092986609788681823 ◽

2009 ◽

Vol 16 (7) ◽

pp. 779-785 ◽

Cited By ~ 4

Author(s):

A.T. Da Poian ◽

F. Carneiro ◽

F. Stauffer

Keyword(s):

Membrane Fusion ◽

Viral Inactivation ◽

Viral Vaccines

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A Virion-Based Assay for Glycoprotein Thermostability Reveals Key Determinants of Filovirus Entry and Its Inhibition

Journal of Virology ◽

10.1128/jvi.00336-20 ◽

2020 ◽

Vol 94 (18) ◽

Cited By ~ 1

Author(s):

Robert H. Bortz ◽

Anthony C. Wong ◽

Michael G. Grodus ◽

Hannah S. Recht ◽

Marc C. Pulanco ◽

...

Keyword(s):

Membrane Fusion ◽

Small Molecule ◽

Viral Entry ◽

Ebola Virus ◽

Elevated Temperatures ◽

Vaccine Development ◽

Cysteine Proteases ◽

Enzyme Linked Immunosorbent Assay ◽

Temperature Dependent ◽

Acid Ph

ABSTRACT Ebola virus (EBOV) entry into cells is mediated by its spike glycoprotein (GP). Following attachment and internalization, virions traffic to late endosomes where GP is cleaved by host cysteine proteases. Cleaved GP then binds its cellular receptor, Niemann-Pick C1. In response to an unknown cellular trigger, GP undergoes conformational rearrangements that drive fusion of viral and endosomal membranes. The temperature-dependent stability (thermostability) of the prefusion conformers of class I viral fusion glycoproteins, including those of filovirus GPs, has provided insights into their propensity to undergo fusion-related rearrangements. However, previously described assays have relied on soluble glycoprotein ectodomains. Here, we developed a simple enzyme-linked immunosorbent assay (ELISA)-based assay that uses the temperature-dependent loss of conformational epitopes to measure thermostability of GP embedded in viral membranes. The base and glycan cap subdomains of all filovirus GPs tested suffered a concerted loss of prefusion conformation at elevated temperatures but did so at different temperature ranges, indicating virus-specific differences in thermostability. Despite these differences, all of these GPs displayed reduced thermostability upon cleavage to GP conformers (GPCL). Surprisingly, acid pH enhanced, rather than decreased, GP thermostability, suggesting it could enhance viral survival in hostile endo/lysosomal compartments. Finally, we confirmed and extended previous findings that some small-molecule inhibitors of filovirus entry destabilize EBOV GP and uncovered evidence that the most potent inhibitors act through multiple mechanisms. We establish the epitope-loss ELISA as a useful tool for studies of filovirus entry, engineering of GP variants with enhanced stability for use in vaccine development, and discovery of new stability-modulating antivirals. IMPORTANCE The development of Ebola virus countermeasures is challenged by our limited understanding of cell entry, especially at the step of membrane fusion. The surface-exposed viral protein, GP, mediates membrane fusion and undergoes major structural rearrangements during this process. The stability of GP at elevated temperatures (thermostability) can provide insights into its capacity to undergo these rearrangements. Here, we describe a new assay that uses GP-specific antibodies to measure GP thermostability under a variety of conditions relevant to viral entry. We show that proteolytic cleavage and acid pH have significant effects on GP thermostability that shed light on their respective roles in viral entry. We also show that the assay can be used to study how small-molecule entry inhibitors affect GP stability. This work provides a simple and readily accessible assay to engineer stabilized GP variants for antiviral vaccines and to discover and improve drugs that act by modulating GP stability.

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Structural impact on SARS-CoV-2 spike protein by D614G substitution

Science ◽

10.1126/science.abf2303 ◽

2021 ◽

pp. eabf2303

Author(s):

Jun Zhang ◽

Yongfei Cai ◽

Tianshu Xiao ◽

Jianming Lu ◽

Hanqin Peng ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Membrane Fusion ◽

Receptor Binding ◽

Viral Entry ◽

Vaccine Development ◽

Spike Protein ◽

Receptor Binding Domain ◽

Structural Rearrangements ◽

Viral Spread ◽

Structural Impact

Substitution for aspartic acid by glycine at position 614 in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 appears to facilitate rapid viral spread. The G614 strain and its recent variants are now the dominant circulating forms. We report here cryo-EM structures of a full-length G614 S trimer, which adopts three distinct prefusion conformations differing primarily by the position of one receptor-binding domain. A loop disordered in the D614 S trimer wedges between domains within a protomer in the G614 spike. This added interaction appears to prevent premature dissociation of the G614 trimer, effectively increasing the number of functional spikes and enhancing infectivity, and to modulate structural rearrangements for membrane fusion. These findings extend our understanding of viral entry and suggest an improved immunogen for vaccine development.

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A novel domain within the CIL regulates egress of IFITM3 from the Golgi and prevents its deleterious accumulation in this apparatus

10.1101/2021.03.29.437470 ◽

2021 ◽

Author(s):

Li Zhong ◽

Rustem Uzbekov ◽

Chloe Journo ◽

Philippe Roingeard ◽

Andrea CIMARELLI

Keyword(s):

Membrane Fusion ◽

Vesicle Trafficking ◽

Transmembrane Proteins ◽

Cellular Membrane ◽

Paroxysmal Kinesigenic Dyskinesia ◽

Fusion Inhibition ◽

Viral Inhibitors

The InterFeron-Induced TransMembrane proteins (IFITMs) are broad viral inhibitors that protect cells by preventing viral-to-cellular membrane fusion and they belong to the dispanin/CD225 family that includes vesicle trafficking regulators and proteins of unknown functions into four subfamilies (A-D). In this study, we uncover a novel domain that regulates the egress of IFITM3 from the Golgi and that is required to prevent IFITM3-driven v- to t-SNAREs membrane fusion inhibition and Golgi dysfunctions. The S-x-K-x-R-D domain is conserved among vertebrate members of the dispanin/CD225 A subfamily that regroups all IFITMs and through the study of mutations identified in patients affected by paroxysmal kinesigenic dyskinesia (PKD), we determine that it is functionally conserved also in PRRT2, member of the B subfamily. Overall, our study defines a novel domain that regulates the egress of dispanin/CD225 members from the Golgi and stresses the importance that regulation of this process bears to preserve the functions of this apparatus.

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Domains and Functions of Spike Protein in SARS-Cov-2 in the Context of Vaccine Design

Viruses ◽

10.3390/v13010109 ◽

2021 ◽

Vol 13 (1) ◽

pp. 109

Author(s):

Xuhua Xia

Keyword(s):

Membrane Fusion ◽

Receptor Binding ◽

Viral Entry ◽

Vaccine Development ◽

Cell Entry ◽

Spike Protein ◽

Structural Domains ◽

Theoretical Approaches ◽

A Cell ◽

And Function

The spike protein in SARS-CoV-2 (SARS-2-S) interacts with the human ACE2 receptor to gain entry into a cell to initiate infection. Both Pfizer/BioNTech’s BNT162b2 and Moderna’s mRNA-1273 vaccine candidates are based on stabilized mRNA encoding prefusion SARS-2-S that can be produced after the mRNA is delivered into the human cell and translated. SARS-2-S is cleaved into S1 and S2 subunits, with S1 serving the function of receptor-binding and S2 serving the function of membrane fusion. Here, I dissect in detail the various domains of SARS-2-S and their functions discovered through a variety of different experimental and theoretical approaches to build a foundation for a comprehensive mechanistic understanding of how SARS-2-S works to achieve its function of mediating cell entry and subsequent cell-to-cell transmission. The integration of structure and function of SARS-2-S in this review should enhance our understanding of the dynamic processes involving receptor binding, multiple cleavage events, membrane fusion, viral entry, as well as the emergence of new viral variants. I highlighted the relevance of structural domains and dynamics to vaccine development, and discussed reasons for the spike protein to be frequently featured in the conspiracy theory claiming that SARS-CoV-2 is artificially created.

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Influence of Respiratory Syncytial Virus F Glycoprotein Conformation on Induction of Protective Immune Responses

Journal of Virology ◽

10.1128/jvi.00338-16 ◽

2016 ◽

Vol 90 (11) ◽

pp. 5485-5498 ◽

Cited By ~ 22

Author(s):

Concepción Palomo ◽

Vicente Mas ◽

Michelle Thom ◽

Mónica Vázquez ◽

Olga Cano ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Membrane Fusion ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Protective Antigen ◽

Protective Efficacy ◽

Viral Fusion ◽

Virus Challenge ◽

Advantages And Disadvantages ◽

Syncytial Virus

ABSTRACTHuman respiratory syncytial virus (hRSV) vaccine development has received new impetus from structure-based studies of its main protective antigen, the fusion (F) glycoprotein. Three soluble forms of F have been described: monomeric, trimeric prefusion, and trimeric postfusion. Most human neutralizing antibodies recognize epitopes found exclusively in prefusion F. Although prefusion F induces higher levels of neutralizing antibodies than does postfusion F, postfusion F can also induce protection against virus challenge in animals. However, the immunogenicity and protective efficacy of the three forms of F have not hitherto been directly compared. Hence, BALB/c mice were immunized with a single dose of the three proteins adjuvanted with CpG and challenged 4 weeks later with virus. Serum antibodies, lung virus titers, weight loss, and pulmonary pathology were evaluated after challenge. Whereas small amounts of postfusion F were sufficient to protect mice, larger amounts of monomeric and prefusion F proteins were required for protection. However, postfusion and monomeric F proteins were associated with more pathology after challenge than was prefusion F. Antibodies induced by all doses of prefusion F, in contrast to other F protein forms, reacted predominantly with the prefusion F conformation. At high doses, prefusion F also induced the highest titers of neutralizing antibodies, and all mice were protected, yet at low doses of the immunogen, these antibodies neutralized virus poorly, and mice were not protected. These findings should be considered when developing new hRSV vaccine candidates.IMPORTANCEProtection against hRSV infection is afforded mainly by neutralizing antibodies, which recognize mostly epitopes found exclusively in the viral fusion (F) glycoprotein trimer, folded in its prefusion conformation, i.e., before activation for membrane fusion. Although prefusion F is able to induce high levels of neutralizing antibodies, highly stable postfusion F (found after membrane fusion) is also able to induce neutralizing antibodies and protect against infection. In addition, a monomeric form of hRSV F that shares epitopes with prefusion F was recently reported. Since each of the indicated forms of hRSV F may have advantages and disadvantages for the development of safe and efficacious subunit vaccines, a direct comparison of the immunogenic properties and protective efficacies of the different forms of hRSV F was made in a mouse model. The results obtained show important differences between the noted immunogens that should be borne in mind when considering the development of hRSV vaccines.

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Evidence That the Transition of HIV-1 Gp41 into a Six-Helix Bundle, Not the Bundle Configuration, Induces Membrane Fusion

The Journal of Cell Biology ◽

10.1083/jcb.151.2.413 ◽

2000 ◽

Vol 151 (2) ◽

pp. 413-424 ◽

Cited By ~ 380

Author(s):

Grigory B. Melikyan ◽

Ruben M. Markosyan ◽

Hila Hemmati ◽

Mary K. Delmedico ◽

Dennis M. Lambert ◽

...

Keyword(s):

Membrane Fusion ◽

Vaccine Development ◽

Intermediate Stage ◽

Viral Fusion ◽

Helix Bundle ◽

Inhibitory Peptides ◽

Rate Limiting Step ◽

Heptad Repeats ◽

Rate Limiting ◽

Hiv 1

Many viral fusion proteins exhibit a six-helix bundle as a core structure. HIV Env–induced fusion was studied to resolve whether membrane merger was due to the transition into the bundle configuration or occurred after bundle formation. Suboptimal temperature was used to arrest fusion at an intermediate stage. When bundle formation was prevented by adding inhibitory peptides at this stage, membranes did not merge upon raising temperature. Inversely, when membrane merger was prevented by incorporating lysophosphatidylcholine (LPC) into cell membranes at the intermediate, the bundle did not form upon optimizing temperature. In the absence of LPC, the six-helix bundle did not form when the temperature of the intermediate was raised for times too short to promote fusion. Kinetic measures showed that after the temperature pulse, cells had not advanced further toward fusion. The latter results indicate that bundle formation is the rate-limiting step between the arrested intermediate and fusion. Electrical measures showed that the HIV Env–induced pore is initially large and grows rapidly. It is proposed that bundle formation and fusion are each contingent on the other and that movement of Env during its transition into the six-helix bundle directly induces the lipid rearrangements of membrane fusion. Because peptide inhibition showed that, at the intermediate stage, the heptad repeats of gp41 have become stably exposed, creation of the intermediate could be of importance in drug and/or vaccine development.

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Analysis of insertion mutants in distal α9 portion of C-terminal heptad repeat (CHR) of HIV-1 gp41 subunit

10.1101/2020.03.30.017129 ◽

2020 ◽

Author(s):

Hongyun Wang ◽

Jiping Song ◽

Yasushi Kawaguchi ◽

Jun-ichiro Inoue ◽

Zene Matsuda

Keyword(s):

Membrane Fusion ◽

Underlying Mechanism ◽

Heptad Repeat ◽

Target Cells ◽

Wild Type ◽

Helix Formation ◽

Fusion Inhibition ◽

The Stability ◽

Α Helix ◽

Insertion Mutants

AbstractWe have made insertion mutants in α9 of HXB2 gp41 and observed similar phenotypes like recent JRFL mutants: insertion of alanine (653+A), but not glutamine (653+Q), severely attenuated membrane fusion. To understand the underlying mechanism, we performed the fusion inhibition assay by corresponding mutant C34 peptides. Both mutant C34 peptides added at the beginning of the coculture of the effector and target cells showed less efficient inhibition of membrane fusion, which was similar to wildtype C34 added after 30 min of coculture, indicating slow association of mutant C34 peptides with the N-terminal heptad region of gp41. Due to uninterpretable CD profiles of C34 and N36, we tested the longer peptide pairs (N46 and C42) and observed CD profiles indicative of weak α-helix formation. The melting temperatures for N46-C42 pairs of 653+A, 653+Q, and wild type were 56.8 °C, 59.8°C, and 96°C, respectively. Taken together, our data suggested that the phenotypic difference in membrane fusion between 653+A and 653+Q (or wild type) was not based on the stability of the six-helix bundle (6HB), but due to differences in the kinetics of 6HB formation. Further, we examined additional insertions (E, R, I, and L) at position 653, for which only I and L showed fusion recovery similar to Q, suggesting that the polar nature of glutamine was not a phenotypic determinant.

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Localization of the Interaction Site of Herpes Simplex Virus Glycoprotein D (gD) on the Membrane Fusion Regulator, gH/gL

Journal of Virology ◽

10.1128/jvi.00983-20 ◽

2020 ◽

Vol 94 (20) ◽

Author(s):

Tina M. Cairns ◽

Doina Atanasiu ◽

Wan Ting Saw ◽

Huan Lou ◽

J. Charles Whitbeck ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Membrane Fusion ◽

Protein Interactions ◽

Vaccine Development ◽

Receptor Interaction ◽

Receptor Binding Site ◽

Interaction Site ◽

Herpes Simplex Virus Glycoprotein ◽

Simplex Virus

ABSTRACT A cascade of protein-protein interactions between four herpes simplex virus (HSV) glycoproteins (gD, gH/gL, and gB) drive fusion between the HSV envelope and host membrane, thereby allowing for virus entry and infection. Specifically, binding of gD to one of its receptors induces a conformational change that allows gD to bind to the regulatory complex gH/gL, which then activates the fusogen gB, resulting in membrane fusion. Using surface plasmon resonance and a panel of anti-gD monoclonal antibodies (MAbs) that sterically blocked the interaction, we previously showed that gH/gL binds directly to gD at sites distinct from the gD receptor binding site. Here, using an analogous strategy, we first evaluated the ability of a panel of uncharacterized anti-gH/gL MAbs to block binding to gD and/or inhibit fusion. We found that the epitopes of four gD-gH/gL-blocking MAbs were located within flexible regions of the gH N terminus and the gL C terminus, while the fifth was placed around gL residue 77. Taken together, our data localized the gD binding region on gH/gL to a group of gH and gL residues at the membrane distal region of the heterodimer. Surprisingly, a second set of MAbs did not block gD-gH/gL binding but instead stabilized the complex by altering the kinetic binding. However, despite this prolonged gD-gH/gL interaction, “stabilizing” MAbs also inhibited cell-cell fusion, suggesting a unique mechanism by which the fusion process is halted. Our findings support targeting the gD-gH/gL interaction to prevent fusion in both therapeutic and vaccine strategies against HSV. IMPORTANCE Key to developing a human HSV vaccine is an understanding of the virion glycoproteins involved in entry. HSV employs multiple glycoproteins for attachment, receptor interaction, and membrane fusion. Determining how these proteins function was resolved, in part, by structural biology coupled with immunological and biologic evidence. After binding, virion gD interacts with a receptor to activate the regulator gH/gL complex, triggering gB to drive fusion. Multiple questions remain, one being the physical location of each glycoprotein interaction site. Using protective antibodies with known epitopes, we documented the long-sought interaction between gD and gH/gL, detailing the region on gD important to create the gD-gH/gL triplex. Now, we have identified the corresponding gD contact sites on gH/gL. Concurrently we discovered a novel mechanism whereby gH/gL antibodies stabilize the complex and inhibit fusion progression. Our model for the gD-gH/gL triplex provides a new framework for studying fusion, which identifies targets for vaccine development.

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